The Amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. Whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. We postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin and both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum (ER) stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.


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Regulatory T cells (Tregs) are a unique subset of CD4 T lymphocytes that have a major role in 30 preventing autoimmunity through regulation of antigen presentation, expression of immunosuppressive 31 molecules, and creating competition for energy and resources [1] . In addition to these canonical functions, 32 non-lymphoid tissue-resident Tregs can also secrete factors that promote tissue regeneration. These 33 specialized tissue-Treg features are instructed by both TCR activation and tissue derived signals, such as 34 IL-33, IL-18, and TGFβ [2][3][4] . Treg mediated tissue repair is driven in part through the secretion of growth 35 factors, such as the epidermal growth factor receptor (EGFR) ligand, amphiregulin (Areg) [2] . In models of 36 tissue injury, Tregs at the tissue site upregulate amphiregulin and induce tissue repair [3][4][5][6] . However, the role 37 of amphiregulin during chronic tissue inflammation and damage has not been fully resolved, as the disease 38 models explored to date largely focus on acute injury models. In one example, an induced model of acute 39 skeletal muscle injury resulted in the accumulation of Tregs expressing amphiregulin within the injured 40 muscle, with stellate stem cells responding to amphiregulin and repopulating myofibrils [5] . In addition, 41 following influenza infection induced damage, Treg-derived amphiregulin acted to repair epithelial cells 42 apart from their immunosuppressive functions [3] .

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Type 1 diabetes (T1D) is a T lymphocyte mediated autoimmune disease that leads to the destruction 44 of insulin producing beta cells within the pancreatic islets of Langerhans. Tregs are critical for protection 45 against autoimmunity and can restrain autoimmune T cells to delay disease progression [7] . We had 46 previously observed that functional Tregs upregulate amphiregulin at the transcriptional level as they enter 47 the pancreatic islets in a non-obese diabetic (NOD) model of type 1 diabetes [8,9] . However, whether the 48 amphiregulin/EGFR axis is engaged and can be protective during autoimmune diabetes was unclear. The 49 potential role for this pathway in T1D is consistent with observed reduced levels of amphiregulin in recently 50 diagnosed patients [10] , and decreased levels of another EGFR ligand, epiregulin, in individuals at a high risk 51 of developing T1D [11] . Furthermore, the EGFR pathway has the potential to be broadly important in the 52 context of T1D, as EGFR has been identified as a susceptibility gene for diabetic neuropathy and diabetes 53 associated cardiac dysfunction [12,13] . We hypothesized, however, that amphiregulin could have a direct role 54 in beta cell survival, based on the observations that beta cells require EGFR signaling for normal 55 development and postnatal proliferation [14] , and that constitutive activation of EGFR on beta cells protects 56 against streptozotocin induced diabetes [15] .

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Here, we report that islet-infiltrating Tregs upregulate amphiregulin in the NOD mouse model of 58 autoimmune diabetes. Moreover, we show that amphiregulin can have a direct effect on beta cells by 59 reducing ER stress in ex vivo cultured islets. However, the deletion of amphiregulin in NOD mice does not 60 change spontaneous autoimmune diabetes development, suggesting that any protective effects induced by 61 amphiregulin are not sufficient to maintain or recover beta cell mass. Therefore, our data suggest that the 62 amphiregulin/EGFR axis is not always effective in controlling inflammatory tissue damage. While 63 potentially promising, the use of amphiregulin as a therapeutic target presents several challenges that will 64 need to be addressed through further study.

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We had previously observed that islet-infiltrating CD4+Foxp3+ Tregs upregulate amphiregulin at 70 the transcriptional level [8,9] . To confirm that amphiregulin was indeed expressed by islet-infiltrating Tregs 71 at the protein level, we used flow cytometric analysis of T cells isolated directly from infiltrated islets or 72 peripheral lymphoid organs and stimulated in vitro. Islet, pancreatic-draining lymph node, and non-draining 73 lymph node T cells from pre-diabetic 14-week-old female NOD mice were analyzed for amphiregulin 74 expression. First, our data show that Foxp3+ Tregs expressed amphiregulin at a greater frequency compared 75 to Foxp3-CD4 Teffs and CD8 Teffs regardless of the tissue site (Figure 1a and 1b). Secondly, among 76 Tregs, the highest frequency of amphiregulin expression was observed within the pancreatic islets (~40%+) 77 ( Figure 1b), suggesting that tissue resident Tregs have an enhanced capacity to express amphiregulin.

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To determine whether Tregs actively produce amphiregulin in vivo, we treated NOD mice with 79 brefeldin A to block amphiregulin secretion, and then analyzed intracellular amphiregulin directly ex vivo.

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Prior models of injury in which Treg derived amphiregulin was essential to tissue recovery showed 91 co-expression of amphiregulin and the IL-33 receptor, ST2 [3] , consistent with the ability for the IL-33/ST2 92 pathway to induce amphiregulin expression in Tregs [16,17] . To determine if islet Tregs similarly expressed 93 ST2, we analyzed ST2 expression on islet CD4 T cells by flow cytometry. In congruence with previous 94 reports [18] , a higher frequency of Tregs expressed ST2 compared to Teffs (Figure 1f). Taken together, these 95 data show that islet resident Tregs express the canonical markers of tissue repair Tregs, amphiregulin and 96 ST2.

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Once we defined Tregs as a major source of amphiregulin within the islets, we analyzed ex vivo 100 expression of the amphiregulin receptor EGFR to determine the potential cellular targets of amphiregulin.

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We observed a relatively high level of EGFR expression on insulin positive beta cells (~73% EGFR+), and 102 beta cell EGFR levels remained consistent between 6 week and 15-17-week-old NOD mice (Figure 2a and 103 2b). To determine whether inflammatory signals during T1D modify levels of EGFR expression on beta 104 cells, we measured EGFR levels on beta cells from NOD.scid mice, which lack T and B cells and do not 105 develop autoimmunity. As there was no difference in EGFR expression between NOD and NOD.scid mice 106 (Figure 2b), we concluded that beta cell EGFR expression is stable regardless of inflammation, which 107 suggested that beta cells could respond to amphiregulin throughout disease.

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In addition to beta cells, a proportion of both CD4+ effector and CD4+Foxp3+ regulatory T cells 109 within the islets expressed EGFR on their cell surface, although Tregs expressed significantly higher levels 110 (~8% compared to ~22%) ( Figure 2c). Therefore, it is possible that Tregs may utilize amphiregulin in a 111 paracrine fashion during autoimmunity, as well as secrete amphiregulin to promote tissue repair [19] .

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Although levels of EGFR expressed on beta cells did not differ between normal and inflammatory 113 conditions, previous studies suggested that EGFR expression on Tregs could be induced in response to 114 inflammation [19] . To determine if EGFR expression is induced on Tregs in response to autoimmune stress, 115 we compared EGFR levels on Tregs in the islets to the levels expressed by Tregs in the non-draining lymph 116 nodes ( Figure 2d). Tregs in the islets indeed showed higher levels of EGFR expression compared to the 117 non-draining lymph nodes, indicating that Tregs upregulate EGFR in response to inflammation within the 118 islets and thus have a better potential to respond to amphiregulin.

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The high demand for insulin production in beta cells results in transient endoplasmic reticulum 123 (ER) stress in response to glucose stimulation [20] . However, under chronic ER stress conditions, such as 124 those found during diabetes, the unfolded protein response (UPR) may lead to loss of beta cell function, 125 and further unresolved ER stress can lead to beta cell death [21,22] . Importantly, amphiregulin was previously 126 shown to be upregulated during beta cell recovery phase after transient hyperglycemia induced by deletion 127 of the UPR sensor IRE1α [23] , suggesting a role for amphiregulin in modulating beta cell mass in response 128 to ER stress. Thus, we asked if amphiregulin was directly capable of reducing ER stress in beta cells.

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Using thapsigargin, a SERCA pump inhibitor, we induced ER stress in cultured NOD.scid islets, 130 and measured transcription of genes commonly associated with the three arms of the UPR (Figure 3).

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Insulin transcript served as a control for similar beta cell distribution among the treatment groups. All of 132 the assessed ER stress genes were upregulated in response to thapsigargin treatment, as expected. ER 133 stressed islets were treated with recombinant amphiregulin protein to assess the potential for amphiregulin 134 to modulate thapsigargin induced ER stress. Early downstream targets of the UPR (sXBP1, ATF4, and 135 ATF6) were unchanged with the addition of amphiregulin, although sXBP1 and ATF6 did trend towards 136 decreased transcription levels. In addition, transcription of the pro-apoptotic factor, CHOP, was unaltered 137 by amphiregulin. However, transcription levels of the chaperone binding immunoglobin protein (BiP) 138 (Grp78) were significantly downregulated with amphiregulin, showing that amphiregulin can directly act 139 upon beta cells. While this evidence suggests that amphiregulin cannot completely prevent or reduce ER 140 stress in ex vivo cultured islets, amphiregulin may still be able to moderate certain aspects of the UPR. As and pertussis toxin as described previously [25] . Similar to our results obtained from NOD diabetes model, 176 amphiregulin seemed to have no impact on the development of EAE (Figure 5b). In congruence with others 177 who have suggested that amphiregulin is dispensable for protective Treg function during EAE [26] , we also 178 saw that the severity of EAE disease remained unchanged between Areg +/+ and Areg -/mice.

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Regulatory T cells that infiltrate or reside within tissues are often uniquely adapted to be effective 183 within their environment. In some situations, Tregs with repair functions exhibit a unique repair 184 transcriptional signature, indicating a specialized subpopulation or lineage [27,28] . Based on single-cell 185 transcriptomics, tissue-resident Tregs differentiate from a lymphoid tissue phenotype into subpopulations 186 that have conserved transcriptional programs [29] . Some of these programs involve unique functions such as 187 maintenance of tissue homeostasis as well as damage repair, and include the EGFR ligand amphiregulin.

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Here we show that pancreatic Tregs can express amphiregulin in addition to other known 189 immunosuppressive molecules [1] . Furthermore, islet-resident Tregs express ST2, the receptor for the 190 alarmin cytokine IL-33. The expression of ST2 and amphiregulin on islet-infiltrating Tregs implies that 191 these cells can respond to tissue damage signals as part of a repair program, in accordance with previous 192 findings [3,16,17] . Importantly, we show that beta cells express high levels of the amphiregulin receptor, 193 EGFR, suggesting that beta cells can respond to amphiregulin. In fact, the presence of recombinant 194 amphiregulin during thapsigargin induced ER stress in cultured islets reduced Grp78 (BiP) transcription, a 195 regulator of the unfolded protein response pathway. All of these observations point to amphiregulin as a 196 potentially important mechanism employed by islet-infiltrating Tregs to curb beta cell destruction.
197 Surprisingly, however, amphiregulin deficiency in NOD mice did not significantly alter autoimmune 198 diabetes incidence or progression.

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The lack of impact of amphiregulin deficiency on autoimmune diabetes led us to question whether 200 amphiregulin also fails to protect in other autoimmune models. Using C57BL/6 amphiregulin deficient 201 mice, we saw that loss of amphiregulin did not impact the severity of EAE induced by MOG35-55 peptide 202 immunization. This observation is consistent with a recent study showing that amphiregulin knock-down 203 in Treg adoptive therapy does not impede their ability to suppress EAE [26] . The protective role of 204 amphiregulin seems to be disease or context dependent, as amphiregulin is effective in controlling immune 205 responses during autoimmune colitis [19] and pristane-induced lupus nephritis [30] .

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The effectiveness of amphiregulin in tissue repair may be dependent on the target cell. In mice, 207 adult beta cells show a relatively low proliferative capacity [31] . This low regenerative potential may limit 208 the ability for amphiregulin to repair damaged beta cells at a rate that can compete with the damage caused 209 by autoimmune assault during T1D. Furthermore, in certain disease models the target of amphiregulin 210 during injury are stem-like cells that undergo induced differentiation into tissue cells to help support tissue 211 repair. For example, amphiregulin expressing Tregs that accumulate in muscle fibers following acute injury 212 prompt the differentiation of muscle satellite cells [5] . Such stem-like pre-cursor cells for beta cells have not 213 been identified. In contrast to acute muscle injury, chronic infection with the parasite Toxoplasma gondii, 214 increases amphiregulin expression in muscle Tregs, but does not prevent tissue damage [32] . In the case of T.

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As the field looks to innovative ways of preventing, attenuating, or reversing T1D disease 219 progression, several methods have become promising. One of these approaches is to attenuate the 220 autoimmune attack on beta cells by reducing the number of activated autoreactive T effectors. Teplizumab 221 -the first FDA approved immunotherapy to delay T1D -is a monoclonal antibody against CD3 that showed 222 increased frequencies of unresponsive CD8 T cells in treated individuals [33] . In addition, current clinical 223 trials are using expanded autologous Tregs re-infused into patients to suppress autoimmune responses [34] .

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However, a second and complementary strategy in T1D therapies is directed towards improving beta cell 225 survival and restoring beta cell mass and function, either through the protection of beta cells against 226 autoimmune attack, or beta cell regeneration. Whether Treg therapy could be harnessed to enhance beta 227 cell survival in addition to inducing autoimmune suppression is unknown, but is an active area of 228 investigation. Apart from endogenous Treg functions, cells could be enhanced to achieve additional 229 functional competency. For example, Tregs could be re-engineered in order to increase antigen specificity 230 either through exogenous CAR or TCR [35] . Treg expression of amphiregulin can be driven by alarmin 231 cytokines (i.e. IL-33 and IL-18) that are produced by injured tissues [3,4,36] . Consistent with the role of IL-232 33, tissue resident Tregs often have high expression of the IL-33 receptor, ST2 [37] . A recent study has 233 overexpressed ST2 on human Tregs to successfully induce TCR-independent reparative mechanisms, in 234 particular increasing the production of amphiregulin [17] . Furthermore, treatment of T1D patient PBMCs in 235 vitro with IL-33 increased Treg frequencies and FOXP3 expression [38] , consistent with previous reports 236 indicating an ability of IL-33 to enhance Treg induction [39] . Thus, with these new technologies emerging, it 237 is of great interest to understand whether the IL-33/amphiregulin/EGFR axis can act directly or indirectly 238 to retain beta cell function during T1D, and whether amphiregulin should be further pursued as a therapeutic 239 target for T1D.

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During autoimmune diseases such as T1D, many tissue resident Tregs are antigen specific [40] and 241 express markers associated with enhanced immunosuppressive functions [41] ; however, it is unclear whether 242 pancreatic Tregs also have a non-immunological purpose within the islets. Our findings provide evidence 243 for a population of islet-infiltrating Tregs that express amphiregulin and ST2; markers associated with tissue 244 repair Tregs. However, while amphiregulin can mitigate beta cell ER stress, analysis of NOD amphiregulin 245 deficient mice suggest that amphiregulin does not overtly impact autoimmune diabetes incidence. Why the 246 amphiregulin/EGFR pathway is not effective in supporting beta cell function is unclear, but could point to 247 another reason for the inability of Tregs to effectively control anti-beta cell responses. As our study did not 248 investigate the outcomes of other tissues often damaged by hyperglycemia (kidneys, blood vessels, heart), 249 amphiregulin could have an effect in improving other comorbidities commonly associated with T1D [13] .

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Lastly, there are several limitations of our study that should be considered before we rule out 251 amphiregulin as an effective pro-beta cell factor. The first being that the effects of amphiregulin during 252 autoimmunity were studied using mice that have a global deletion of amphiregulin. The amphiregulin 253 mutation may have resulted in activation of compensatory mechanisms, for example through upregulation 254 of other EGFR ligands. Alternatively, Tregs are known to compensate for lost regulatory functions via 255 upregulation of alternative suppressive mechanisms [42] . Secondly, although islet Tregs produce 256 amphiregulin and beta cells express EGFR, we did not investigate whether EGFR is actively signaling in 257 beta cells. It is possible that post-translational processing of amphiregulin, or downstream EGFR signaling 258 is changed during T1D leading to sub-optimal amphiregulin/EGFR axis activation. Additionally, this study 259 focused on the effect of amphiregulin in pancreatic islets during autoimmune diabetes and did not explore

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In vivo cytokine quantification -In vivo cytokine production was evaluated as described previously [44] .

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Briefly, Brefeldin A (BFA) powder was dissolved in DMSO (20mg/mL) and stored at -20°C. Immediately 294 before use, concentrated BFA stock was diluted to a working concentration (0.5mg/mL) in PBS and 500 295 uL was injected via tail vein. Six hours post injection, mice were euthanized, and islets and lymph nodes 296 were analyzed by flow cytometry on Cytek Aurora (Cytek Biosciences).

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In vitro T cell activation -Islets from 14-week-old female NOD mice were isolated and stimulated at 37°

Figure 3
Amphiregulin reduces beta cell ER stress. qPCR analysis of cultured islets treated with thapsigargin. Islets from four NOD.scid mice were pooled and either cultured in 10% FBS RPMI with or without 100ng/mL recombinant mouse Areg and/or 5mM Thapsigargin for 7 hours. Expression relative to Gapdh. One-way ANOVA with Tukey multiple comparison. N=3, Data shown as Mean with SEM.